电泳法筛选酶及其抑制剂和大管电泳研究
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摘要
药物筛选是当今医药领域新兴的课题,这一课题具有极高的临床价值和深远的社会意义,因此,这一研究领域的更新和发展一直倍受瞩目。近年来,诸多研究的目光投向筛选方法的改进与更新上以提高筛选的效率和准确性,进而应对数目迅猛增多的候选药用化合物。毛细管电泳技术以其高效、低耗、快速和易自动化等诸多特点,自一出现就引起人们的高度重视,至今在许多领域依然发挥着不可替代的作用。特别是毛细管电泳技术的高效分离机制,可以弥补传统药物筛选中的一些不足,因此,在一定程度上提高了结果的准确性,增加了可信度。另一方面,酶作为具有生物活性的催化剂与人类机体的健康息息相关,近年来,通过选用相关酶的抑制剂对某些疾病进行治疗的方法十分有效,这也为新药的发现和筛选提供了一条新思路。本论文以毛细管电泳技术为基础、结合高效的激光诱导荧光检测方法,选用酪氨酸蛋白激酶、信号肽酶为探针酶,分别系统地对其活性及其抑制剂的效果进行了考察,结果证明毛细管电泳法在该领域的可操作性及独具的优势。
在利用和发挥传统毛细管电泳技术优势的同时,不可避免的会受到其载样量较低的缺点的限制,尤其是在应用于多维分析中时,过低载样量带来不可逾越的困难。本文在传统毛细管电泳结构的基础上引入内制冷体系,借此增加有效分离通道的空间实现载样量的提升。在前期实验证实该装置的初步可操作性后,本文引入以电喷雾法自制的氨丙基纳米硅球作为电泳缓冲液添加剂,以一组芳香酸为模型化合物,实现对该装置效率提高的目的,进而证实该装置在药物研究领域可发挥的潜在作用。
通过本文系统的论证,传统毛细管电泳法在药物筛选领域具有很强的可操作性,能够为药物筛选及高通量筛选方法的更新和改进做出相应的贡献;同时本文对新型大管电泳装置的优化,为广义的电泳技术提供了更为广博的适用空间,为电泳技术的发展提供了基础的理论论据。
As being a developing project, drug screening has been attracted increased attention since coming into being due to its both clinic value and social significance. In recent years, more interests are focusing on the improvement of efficiency and accuracy of screening methods to deal with the plenty of potential drug candidates. Capillary electrophoresis (CE) is widely perceived as a fast, economical, and efficient separation technique and especially, when used in combination with laser-induced fluorescence (LIF) detection, has high sensitivity. This separation-based technique can compensate the shortage of some traditional drug screening methods. On the other hand, the study of novel candidates which specially target the special enzyme is an important aspect in the pharmaceutical industry. Herein, we present a robust CE/LIF method, using tyrosine kinase and signal peptides as probe enzymes, and systematically proved the feasibility of CE method performing the characterization and inhibitor screen.
Meanwhile, some recent improvements have been reported for enhancing the loading capacity of CE-based methods, however, loading capacity is still a critical issue if CE approaches are to be employed in multi-dimensional separation approaches. Based on previous reports, a novel wide-bore (WE) electrophoresis method employing nanoparticles as a pseudostationary phase was evaluated for improving separation efficiency using a model mixture of six aromatic acids. The nanoparticles used in this work were prepared in-house by an electrospray method. The results indicted that the sample loading capacity and resolution were both significantly increased by utilizing the novel WE approach in conjunction with aminopropyl modified nanoparticles as an additive in running buffer.
In summary, traditional CE methods is an operational method which may contribute to the improvement of drug screen and high throughput screen methods. And the exploration and optimization of novel wide-bore electrophoresis enhanced the practicability and widen the field of electrophoresis techinique.
在利用和发挥传统毛细管电泳技术优势的同时,不可避免的会受到其载样量较低的缺点的限制,尤其是在应用于多维分析中时,过低载样量带来不可逾越的困难。本文在传统毛细管电泳结构的基础上引入内制冷体系,借此增加有效分离通道的空间实现载样量的提升。在前期实验证实该装置的初步可操作性后,本文引入以电喷雾法自制的氨丙基纳米硅球作为电泳缓冲液添加剂,以一组芳香酸为模型化合物,实现对该装置效率提高的目的,进而证实该装置在药物研究领域可发挥的潜在作用。
通过本文系统的论证,传统毛细管电泳法在药物筛选领域具有很强的可操作性,能够为药物筛选及高通量筛选方法的更新和改进做出相应的贡献;同时本文对新型大管电泳装置的优化,为广义的电泳技术提供了更为广博的适用空间,为电泳技术的发展提供了基础的理论论据。
As being a developing project, drug screening has been attracted increased attention since coming into being due to its both clinic value and social significance. In recent years, more interests are focusing on the improvement of efficiency and accuracy of screening methods to deal with the plenty of potential drug candidates. Capillary electrophoresis (CE) is widely perceived as a fast, economical, and efficient separation technique and especially, when used in combination with laser-induced fluorescence (LIF) detection, has high sensitivity. This separation-based technique can compensate the shortage of some traditional drug screening methods. On the other hand, the study of novel candidates which specially target the special enzyme is an important aspect in the pharmaceutical industry. Herein, we present a robust CE/LIF method, using tyrosine kinase and signal peptides as probe enzymes, and systematically proved the feasibility of CE method performing the characterization and inhibitor screen.
Meanwhile, some recent improvements have been reported for enhancing the loading capacity of CE-based methods, however, loading capacity is still a critical issue if CE approaches are to be employed in multi-dimensional separation approaches. Based on previous reports, a novel wide-bore (WE) electrophoresis method employing nanoparticles as a pseudostationary phase was evaluated for improving separation efficiency using a model mixture of six aromatic acids. The nanoparticles used in this work were prepared in-house by an electrospray method. The results indicted that the sample loading capacity and resolution were both significantly increased by utilizing the novel WE approach in conjunction with aminopropyl modified nanoparticles as an additive in running buffer.
In summary, traditional CE methods is an operational method which may contribute to the improvement of drug screen and high throughput screen methods. And the exploration and optimization of novel wide-bore electrophoresis enhanced the practicability and widen the field of electrophoresis techinique.
引文
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